Re: [ccp4bb] Compound with flexible conformation but nM Kd

[IGBMC]

Le Vendredi 27 Avril 2018 14:51 CEST, vincent Chaptal  
a écrit:

Just a little remark (I hope I'm not splitting hairs): it is not exact that the 
alkyl chain "creates a lot of entropy". Instead the mark of dissolution of 
hydrophobic compounds in aqueous solvent is a strong decrease of entropy due to 
water molecules becoming ordered around the alkyl chain. This is precisely due 
to this  unfavorable entropic term that hydrophobic compounds do not dissolve 
in water, not because of an unfavorable enthalpic term.
Philippe D


> Dear Wenhe,
>
> A thought came to mind after having read all the other threads, for

> which I generally agree.
> An alkyl chain on a molecule (charged? hydrophilic?, you mention a
> negatively charged binding site) will most likely not lead to micelle
> formation as the cmc of the object will be most likely higher than the
> amount you use in solution, especially at uM concentrations. But the 
> alkyl chain nevertheless creates a lot of entropy, it doesn't like being
> in the water. How long is the alky chain? if it is 8-9-10 or even 11 
> carbons, it is likely to be not hydrophobic enough to want to burry the
> side chains into a micelle, and be very exchangeable in solution, yet
> not happy to be there. Binding onto a surface would reduce entropy,

> resulting in a better kon?
> You could try ITC, you will have access to detlaH and deltaG of binding,
> and by comparing with your other molecules maybe something would come up?
>
> please correct me if I'm wrong.
>
> All the best
> Vincent
>
> On 27/04/2018 05:07, WENHE ZHONG wrote:
> > Hi Philippe,
> >
> > The affinity was measured by SPR where we immobilized the protein on
> > the chip. One thing I forgot to mention is that the association rate
> > (kon) shown in SPR experiment for this compound is faster (>10-fold
> > faster) compared to other analogues with similar koff. There is a

> > pi-pi interaction between the scaffold structure and the protein
> > (tyrosine ring). Is it possible that the hydrophobic substituent could
> > facilitate the formation of this pi-pi interaction but not necessary
> > to involve in the interaction? Thanks.
> >
> > Kind regards,
> > Wenhe
> >
> >> On Apr 27, 2018, at 1:50 AM, DUMAS Philippe (IGBMC)
> >> mailto:p.du...@ibmc-cnrs.unistra.fr>>
> >> wrote:
> >>
> >>
> >> Le Jeudi 26 Avril 2018 16:50 CEST, WENHE ZHONG
> >> mailto:wenhezhong.xmu@gmail.com>>
> >> a écrit:
> >>
> >> Just to be sure: how was the nM affinity evaluated ? By in vitro

> >> measurements, or by obtaining an IC50 by tests on cells ?
> >> Of course, if you are mentioning an IC50, you may have a measurement
> >> of the efficacy of drug entrance in the cells, not just of specific
> >> binding to your protein target.
> >> Philippe D.
> >>
> >>> Dear Community,
> >>>
> >>> A little bit out of topic here. We are applying the structure-based
> >>> approach to design compounds that can bind our protein target. We
> >>> have synthesized a series of analogues based on the same scaffold
> >>> with different substituents at one particular site. The most potent
> >>> analogue (nM Kd) has a long alkyl chain substituent. We thought this
> >>> hydrophobic substituent should have strong interactions with the 
> >>> target protein leading to nM range affinity. However, crystal
> >>> structures show very weak densities for this substituent and no

> >>> obvious interaction between the substituent and the target protein,
> >>> suggesting that this long alkyl chain substituent is flexible
> >>> without binding to the protein. This binding site is relatively

> >>> negative charged according to the electrostatic potential analysis.
> >>>
> >>> So it is a puzzle to me that how this dynamic and hydrophobic alkyl
> >>> chain substituent can lead the compound to achieve nM affinity
> >>> (>10-fold better than any other substituent) — in particular the
> >>> binding site is not hydrophobic and no interaction is found between
> >>> the substituent and the protein.
> >>>
> >>> Anything I have miss here that can increase the binding affinity 
> >>> without interacting with the target?
> >>>
> >>> Thanks.
> >>>
> >>> Kind regards,
> >>> Wenhe
> >>>
> >>>
> >>>
> >>
> >>
> >>
> >>
> >>
> >>
> >
>
> --
>
> Vincent Chaptal, PhD
>
> Institut de Biologie et Chimie des Protéines
>
> Drug Resistance and Membrane Proteins Laboratory
>
> 7 passage du Vercors
>
> 69007 LYON
>
> FRANCE
>
> +33 4 37 65 29 01
>
> http://www.ibcp.fr
>
>

Re: [ccp4bb] Compound with flexible conformation but nM Kd

[Wenhe Zhong]

Thanks for the thoughtful thought. The alkyl chain has 8 carbons. The scaffold 
that the alkyl chain attached to has 3 hydrogen bonds donors and 3 H-bond 
acceptors and with one phenyl ring. The overall cLogP is around 4 — slightly 
higher than the Lipinski's rule of five. Yes, I have planed to run the ITC to 
see what information I can get.  

Wenhe

> On 27 Apr 2018, at 8:51 pm, vincent Chaptal  wrote:
> 
> Dear Wenhe, 
> 
> A thought came to mind after having read all the other threads, for which I 
> generally agree. 
> An alkyl chain on a molecule (charged? hydrophilic?, you mention a negatively 
> charged binding site) will most likely not lead to micelle formation as the 
> cmc of the object will be most likely higher than the amount you use in 
> solution, especially at uM concentrations. But the alkyl chain nevertheless 
> creates a lot of entropy, it doesn't like being in the water. How long is the 
> alky chain? if it is 8-9-10 or even 11 carbons, it is likely to be not 
> hydrophobic enough to want to burry the side chains into a micelle, and be 
> very exchangeable in solution, yet not happy to be there. Binding onto a 
> surface would reduce entropy, resulting in a better kon? 
> You could try ITC, you will have access to detlaH and deltaG of binding, and 
> by comparing with your other molecules maybe something would come up? 
> 
> please correct me if I'm wrong. 
> 
> All the best
> Vincent
> 
> On 27/04/2018 05:07, WENHE ZHONG wrote:
>> Hi Philippe,
>> 
>> The affinity was measured by SPR where we immobilized the protein on the 
>> chip. One thing I forgot to mention is that the association rate (kon) shown 
>> in SPR experiment for this compound is faster (>10-fold faster) compared to 
>> other analogues with similar koff. There is a pi-pi interaction between the 
>> scaffold structure and the protein (tyrosine ring). Is it possible that the 
>> hydrophobic substituent could facilitate the formation of this pi-pi 
>> interaction but not necessary to involve in the interaction? Thanks.
>> 
>> Kind regards,
>> Wenhe
>> 
>>> On Apr 27, 2018, at 1:50 AM, DUMAS Philippe (IGBMC) 
>>> mailto:p.du...@ibmc-cnrs.unistra.fr>> wrote:
>>> 
>>> 
>>> Le Jeudi 26 Avril 2018 16:50 CEST, WENHE ZHONG 
>>> mailto:wenhezhong.xmu@gmail.com>> a 
>>> écrit:
>>> 
>>> Just to be sure: how was the nM affinity evaluated ? By in vitro 
>>> measurements, or by obtaining an IC50 by tests on cells ?
>>> Of course, if you are mentioning an IC50, you may have a measurement of the 
>>> efficacy of drug entrance in the cells, not just of specific binding to 
>>> your protein target.
>>> Philippe D.
>>> 
 Dear Community,
 
 A little bit out of topic here. We are applying the structure-based 
 approach to design compounds that can bind our protein target. We have 
 synthesized a series of analogues based on the same scaffold with 
 different substituents at one particular site. The most potent analogue 
 (nM Kd) has a long alkyl chain substituent. We thought this hydrophobic 
 substituent should have strong interactions with the target protein 
 leading to nM range affinity. However, crystal structures show very weak 
 densities for this substituent and no obvious interaction between the 
 substituent and the target protein, suggesting that this long alkyl chain 
 substituent is flexible without binding to the protein. This binding site 
 is relatively negative charged according to the electrostatic potential 
 analysis.
 
 So it is a puzzle to me that how this dynamic and hydrophobic alkyl chain 
 substituent can lead the compound to achieve nM affinity (>10-fold better 
 than any other substituent) — in particular the binding site is not 
 hydrophobic and no interaction is found between the substituent and the 
 protein.
 
 Anything I have miss here that can increase the binding affinity without 
 interacting with the target?
 
 Thanks.
 
 Kind regards,
 Wenhe
 
 
 
>>> 
>>> 
>>> 
>>> 
>>> 
>>> 
>> 
> 
> -- 
> Vincent Chaptal, PhD
> Institut de Biologie et Chimie des Protéines
> Drug Resistance and Membrane Proteins Laboratory
> 7 passage du Vercors 
> 69007 LYON
> FRANCE
> +33 4 37 65 29 01
> http://www.ibcp.fr 
> 
> 

Re: [ccp4bb] Compound with flexible conformation but nM Kd

[vincent Chaptal]

Dear Wenhe,

A thought came to mind after having read all the other threads, for 
which I generally agree.
An alkyl chain on a molecule (charged? hydrophilic?, you mention a 
negatively charged binding site) will most likely not lead to micelle 
formation as the cmc of the object will be most likely higher than the 
amount you use in solution, especially at uM concentrations. But the 
alkyl chain nevertheless creates a lot of entropy, it doesn't like being 
in the water. How long is the alky chain? if it is 8-9-10 or even 11 
carbons, it is likely to be not hydrophobic enough to want to burry the 
side chains into a micelle, and be very exchangeable in solution, yet 
not happy to be there. Binding onto a surface would reduce entropy, 
resulting in a better kon?
You could try ITC, you will have access to detlaH and deltaG of binding, 
and by comparing with your other molecules maybe something would come up?


please correct me if I'm wrong.

All the best
Vincent

On 27/04/2018 05:07, WENHE ZHONG wrote:

Hi Philippe,

The affinity was measured by SPR where we immobilized the protein on 
the chip. One thing I forgot to mention is that the association rate 
(kon) shown in SPR experiment for this compound is faster (>10-fold 
faster) compared to other analogues with similar koff. There is a 
pi-pi interaction between the scaffold structure and the protein 
(tyrosine ring). Is it possible that the hydrophobic substituent could 
facilitate the formation of this pi-pi interaction but not necessary 
to involve in the interaction? Thanks.


Kind regards,
Wenhe

On Apr 27, 2018, at 1:50 AM, DUMAS Philippe (IGBMC) 
mailto:p.du...@ibmc-cnrs.unistra.fr>> 
wrote:



Le Jeudi 26 Avril 2018 16:50 CEST, WENHE ZHONG 
mailto:wenhezhong.xmu@gmail.com>> 
a écrit:


Just to be sure: how was the nM affinity evaluated ? By in vitro 
measurements, or by obtaining an IC50 by tests on cells ?
Of course, if you are mentioning an IC50, you may have a measurement 
of the efficacy of drug entrance in the cells, not just of specific 
binding to your protein target.

Philippe D.


Dear Community,

A little bit out of topic here. We are applying the structure-based 
approach to design compounds that can bind our protein target. We 
have synthesized a series of analogues based on the same scaffold 
with different substituents at one particular site. The most potent 
analogue (nM Kd) has a long alkyl chain substituent. We thought this 
hydrophobic substituent should have strong interactions with the 
target protein leading to nM range affinity. However, crystal 
structures show very weak densities for this substituent and no 
obvious interaction between the substituent and the target protein, 
suggesting that this long alkyl chain substituent is flexible 
without binding to the protein. This binding site is relatively 
negative charged according to the electrostatic potential analysis.


So it is a puzzle to me that how this dynamic and hydrophobic alkyl 
chain substituent can lead the compound to achieve nM affinity 
(>10-fold better than any other substituent) — in particular the 
binding site is not hydrophobic and no interaction is found between 
the substituent and the protein.


Anything I have miss here that can increase the binding affinity 
without interacting with the target?


Thanks.

Kind regards,
Wenhe














--

Vincent Chaptal, PhD

Institut de Biologie et Chimie des Protéines

Drug Resistance and Membrane Proteins Laboratory

7 passage du Vercors

69007 LYON

FRANCE

+33 4 37 65 29 01

http://www.ibcp.fr

Re: [ccp4bb] Compound with flexible conformation but nM Kd

[Frank von Delft]

Long alkyl chains tend to bad things to hang onto any compound, if it's 
to be biologically useful.  Go consult your friendly local medicinal 
chemist, chances are they'll freak out.



Anyway, long alkyl chains LOVE binding to surfaces, which is of course 
exactly the sort of thing that generates the signal in SPR.  Go consult 
your friendly local SPR ligand-binding expert, chances are they'll point 
to a number of red flags in your readout - not least the fact that it's 
a massive outlier in the chemical series (if I understood your correctly.)



Frank



On 27/04/2018 10:00, Barone, Matthias wrote:


we also do structure-activity relationship and rational drug design. 
And I agree with Christian: never rely on one single method and try to 
include a homogenous assay, such as ITC or FT. you mention a tyrosine 
involved in the binding pocket. Did you try to track the Tyr in FT?


best, matthias


*From:* CCP4 bulletin board  on behalf of 
Christian Roth 

*Sent:* Friday, April 27, 2018 9:00:03 AM
*To:* CCP4BB@JISCMAIL.AC.UK
*Subject:* Re: [ccp4bb] Compound with flexible conformation but nM Kd
Anectodal evidence I have heard from colleagues working with things, 
which are immobilized is that the measured Kd value on the surface can 
be wildly different from what is measured in solution. A superbinder 
on a surface might not be as good in solution. There seems still a lot 
of debate why that is.


Cheers
Christian

On Fri, Apr 27, 2018 at 5:07 AM, WENHE ZHONG 
mailto:wenhezhong.xmu@gmail.com>> 
wrote:


Hi Philippe,

The affinity was measured by SPR where we immobilized the protein
on the chip. One thing I forgot to mention is that the association
rate (kon) shown in SPR experiment for this compound is faster
(>10-fold faster) compared to other analogues with similar koff.
There is a pi-pi interaction between the scaffold structure and
the protein (tyrosine ring). Is it possible that the hydrophobic
substituent could facilitate the formation of this pi-pi
interaction but not necessary to involve in the interaction? Thanks.

Kind regards,
Wenhe


On Apr 27, 2018, at 1:50 AM, DUMAS Philippe (IGBMC)
mailto:p.du...@ibmc-cnrs.unistra.fr>> wrote:


Le Jeudi 26 Avril 2018 16:50 CEST, WENHE ZHONG
mailto:wenhezhong.xmu@gmail.com>> a écrit:

Just to be sure: how was the nM affinity evaluated ? By in vitro
measurements, or by obtaining an IC50 by tests on cells ?
Of course, if you are mentioning an IC50, you may have a
measurement of the efficacy of drug entrance in the cells, not
just of specific binding to your protein target.
Philippe D.


Dear Community,

A little bit out of topic here. We are applying the
structure-based approach to design compounds that can bind our
protein target. We have synthesized a series of analogues based
on the same scaffold with different substituents at one
particular site. The most potent analogue (nM Kd) has a long
alkyl chain substituent. We thought this hydrophobic substituent
should have strong interactions with the target protein leading
to nM range affinity. However, crystal structures show very weak
densities for this substituent and no obvious interaction
between the substituent and the target protein, suggesting that
this long alkyl chain substituent is flexible without binding to
the protein. This binding site is relatively negative charged
according to the electrostatic potential analysis.

So it is a puzzle to me that how this dynamic and hydrophobic
alkyl chain substituent can lead the compound to achieve nM
affinity (>10-fold better than any other substituent) — in
particular the binding site is not hydrophobic and no
interaction is found between the substituent and the protein.

Anything I have miss here that can increase the binding affinity
without interacting with the target?

Thanks.

Kind regards,
Wenhe

Re: [ccp4bb] Compound with flexible conformation but nM Kd

[Barone, Matthias]

we also do structure-activity relationship and rational drug design. And I 
agree with Christian: never rely on one single method and try to include a 
homogenous assay, such as ITC or FT. you mention a tyrosine involved in the 
binding pocket. Did you try to track the Tyr in FT?

best, matthias


From: CCP4 bulletin board  on behalf of Christian Roth 

Sent: Friday, April 27, 2018 9:00:03 AM
To: CCP4BB@JISCMAIL.AC.UK
Subject: Re: [ccp4bb] Compound with flexible conformation but nM Kd

Anectodal evidence I have heard from colleagues working with things, which are 
immobilized is that the measured Kd value on the surface can be wildly 
different from what is measured in solution. A superbinder on a surface might 
not be as good in solution. There seems still a lot of debate why that is.

Cheers
Christian

On Fri, Apr 27, 2018 at 5:07 AM, WENHE ZHONG 
mailto:wenhezhong.xmu@gmail.com>> wrote:
Hi Philippe,

The affinity was measured by SPR where we immobilized the protein on the chip. 
One thing I forgot to mention is that the association rate (kon) shown in SPR 
experiment for this compound is faster (>10-fold faster) compared to other 
analogues with similar koff. There is a pi-pi interaction between the scaffold 
structure and the protein (tyrosine ring). Is it possible that the hydrophobic 
substituent could facilitate the formation of this pi-pi interaction but not 
necessary to involve in the interaction? Thanks.

Kind regards,
Wenhe

On Apr 27, 2018, at 1:50 AM, DUMAS Philippe (IGBMC) 
mailto:p.du...@ibmc-cnrs.unistra.fr>> wrote:


Le Jeudi 26 Avril 2018 16:50 CEST, WENHE ZHONG 
mailto:wenhezhong.xmu@gmail.com>> a écrit:

Just to be sure: how was the nM affinity evaluated ? By in vitro measurements, 
or by obtaining an IC50 by tests on cells ?
Of course, if you are mentioning an IC50, you may have a measurement of the 
efficacy of drug entrance in the cells, not just of specific binding to your 
protein target.
Philippe D.

Dear Community,

A little bit out of topic here. We are applying the structure-based approach to 
design compounds that can bind our protein target. We have synthesized a series 
of analogues based on the same scaffold with different substituents at one 
particular site. The most potent analogue (nM Kd) has a long alkyl chain 
substituent. We thought this hydrophobic substituent should have strong 
interactions with the target protein leading to nM range affinity. However, 
crystal structures show very weak densities for this substituent and no obvious 
interaction between the substituent and the target protein, suggesting that 
this long alkyl chain substituent is flexible without binding to the protein. 
This binding site is relatively negative charged according to the electrostatic 
potential analysis.

So it is a puzzle to me that how this dynamic and hydrophobic alkyl chain 
substituent can lead the compound to achieve nM affinity (>10-fold better than 
any other substituent) — in particular the binding site is not hydrophobic and 
no interaction is found between the substituent and the protein.

Anything I have miss here that can increase the binding affinity without 
interacting with the target?

Thanks.

Kind regards,
Wenhe

Re: [ccp4bb] Compound with flexible conformation but nM Kd

[Christian Roth]

Anectodal evidence I have heard from colleagues working with things, which
are immobilized is that the measured Kd value on the surface can be wildly
different from what is measured in solution. A superbinder on a surface
might not be as good in solution. There seems still a lot of debate why
that is.

Cheers
Christian

On Fri, Apr 27, 2018 at 5:07 AM, WENHE ZHONG 
wrote:

> Hi Philippe,
>
> The affinity was measured by SPR where we immobilized the protein on the
> chip. One thing I forgot to mention is that the association rate (kon)
> shown in SPR experiment for this compound is faster (>10-fold faster)
> compared to other analogues with similar koff. There is a pi-pi
> interaction between the scaffold structure and the protein (tyrosine ring).
> Is it possible that the hydrophobic substituent could facilitate the
> formation of this pi-pi interaction but not necessary to involve in the
> interaction? Thanks.
>
> Kind regards,
> Wenhe
>
> On Apr 27, 2018, at 1:50 AM, DUMAS Philippe (IGBMC) <
> p.du...@ibmc-cnrs.unistra.fr> wrote:
>
>
> Le Jeudi 26 Avril 2018 16:50 CEST, WENHE ZHONG <
> wenhezhong.xmu@gmail.com> a écrit:
>
> Just to be sure: how was the nM affinity evaluated ? By in vitro
> measurements, or by obtaining an IC50 by tests on cells ?
> Of course, if you are mentioning an IC50, you may have a measurement of
> the efficacy of drug entrance in the cells, not just of specific binding to
> your protein target.
> Philippe D.
>
> Dear Community,
>
> A little bit out of topic here. We are applying the structure-based
> approach to design compounds that can bind our protein target. We have
> synthesized a series of analogues based on the same scaffold with different
> substituents at one particular site. The most potent analogue (nM Kd) has a
> long alkyl chain substituent. We thought this hydrophobic substituent
> should have strong interactions with the target protein leading to nM range
> affinity. However, crystal structures show very weak densities for this
> substituent and no obvious interaction between the substituent and the
> target protein, suggesting that this long alkyl chain substituent is
> flexible without binding to the protein. This binding site is relatively
> negative charged according to the electrostatic potential analysis.
>
> So it is a puzzle to me that how this dynamic and hydrophobic alkyl chain
> substituent can lead the compound to achieve nM affinity (>10-fold better
> than any other substituent) — in particular the binding site is not
> hydrophobic and no interaction is found between the substituent and the
> protein.
>
> Anything I have miss here that can increase the binding affinity without
> interacting with the target?
>
> Thanks.
>
> Kind regards,
> Wenhe
>
>
>
>
>
>
>
>
>
>
>

Re: [ccp4bb] Compound with flexible conformation but nM Kd

[WENHE ZHONG]

Hi Philippe,

The affinity was measured by SPR where we immobilized the protein on the chip. 
One thing I forgot to mention is that the association rate (kon) shown in SPR 
experiment for this compound is faster (>10-fold faster) compared to other 
analogues with similar koff. There is a pi-pi interaction between the scaffold 
structure and the protein (tyrosine ring). Is it possible that the hydrophobic 
substituent could facilitate the formation of this pi-pi interaction but not 
necessary to involve in the interaction? Thanks.

Kind regards,
Wenhe

> On Apr 27, 2018, at 1:50 AM, DUMAS Philippe (IGBMC) 
>  wrote:
> 
> 
> Le Jeudi 26 Avril 2018 16:50 CEST, WENHE ZHONG  
> a écrit:
> 
> Just to be sure: how was the nM affinity evaluated ? By in vitro 
> measurements, or by obtaining an IC50 by tests on cells ?
> Of course, if you are mentioning an IC50, you may have a measurement of the 
> efficacy of drug entrance in the cells, not just of specific binding to your 
> protein target.
> Philippe D.
> 
>> Dear Community,
>> 
>> A little bit out of topic here. We are applying the structure-based approach 
>> to design compounds that can bind our protein target. We have synthesized a 
>> series of analogues based on the same scaffold with different substituents 
>> at one particular site. The most potent analogue (nM Kd) has a long alkyl 
>> chain substituent. We thought this hydrophobic substituent should have 
>> strong interactions with the target protein leading to nM range affinity. 
>> However, crystal structures show very weak densities for this substituent 
>> and no obvious interaction between the substituent and the target protein, 
>> suggesting that this long alkyl chain substituent is flexible without 
>> binding to the protein. This binding site is relatively negative charged 
>> according to the electrostatic potential analysis.
>> 
>> So it is a puzzle to me that how this dynamic and hydrophobic alkyl chain 
>> substituent can lead the compound to achieve nM affinity (>10-fold better 
>> than any other substituent) — in particular the binding site is not 
>> hydrophobic and no interaction is found between the substituent and the 
>> protein.
>> 
>> Anything I have miss here that can increase the binding affinity without 
>> interacting with the target?
>> 
>> Thanks.
>> 
>> Kind regards,
>> Wenhe
>> 
>> 
>> 
> 
> 
> 
> 
> 
> 

Re: [ccp4bb] Compound with flexible conformation but nM Kd

[Edward A. Berry]

For proteins in membranes, or proteins purified in the presence of detergents 
and/or lipids, the active site is sometimes surrounded by a hydrophobic mileau. 
The actual concentration that the binding site sees is then dependent on 
partitioning of the ligand between the water (where its concentration for Kd is 
determined) and the hydrophobic phase. Hydrophobic substituents could then 
result in higher concentrations at the binding site, and thus lower the 
apparent Kd, while having nothing to do with binding.

On 04/26/2018 10:50 AM, WENHE ZHONG wrote:

Dear Community,

A little bit out of topic here. We are applying the structure-based approach to 
design compounds that can bind our protein target. We have synthesized a series 
of analogues based on the same scaffold with different substituents at one 
particular site. The most potent analogue (nM Kd) has a long alkyl chain 
substituent. We thought this hydrophobic substituent should have strong 
interactions with the target protein leading to nM range affinity. However, 
crystal structures show very weak densities for this substituent and no obvious 
interaction between the substituent and the target protein, suggesting that 
this long alkyl chain substituent is flexible without binding to the protein. 
This binding site is relatively negative charged according to the electrostatic 
potential analysis.

So it is a puzzle to me that how this dynamic and hydrophobic alkyl chain 
substituent can lead the compound to achieve nM affinity (>10-fold better than 
any other substituent) — in particular the binding site is not hydrophobic and no 
interaction is found between the substituent and the protein.

Anything I have miss here that can increase the binding affinity without 
interacting with the target?

Thanks.

Kind regards,
Wenhe

Re: [ccp4bb] Compound with flexible conformation but nM Kd

[IGBMC]

Le Jeudi 26 Avril 2018 16:50 CEST, WENHE ZHONG  a 
écrit:

Just to be sure: how was the nM affinity evaluated ? By in vitro measurements, 
or by obtaining an IC50 by tests on cells ?
Of course, if you are mentioning an IC50, you may have a measurement of the 
efficacy of drug entrance in the cells, not just of specific binding to your 
protein target.
Philippe D.

> Dear Community,
>
> A little bit out of topic here. We are applying the structure-based approach 
> to design compounds that can bind our protein target. We have synthesized a 
> series of analogues based on the same scaffold with different substituents at 
> one particular site. The most potent analogue (nM Kd) has a long alkyl chain 
> substituent. We thought this hydrophobic substituent should have strong 
> interactions with the target protein leading to nM range affinity. However, 
> crystal structures show very weak densities for this substituent and no 
> obvious interaction between the substituent and the target protein, 
> suggesting that this long alkyl chain substituent is flexible without binding 
> to the protein. This binding site is relatively negative charged according to 
> the electrostatic potential analysis.
>
> So it is a puzzle to me that how this dynamic and hydrophobic alkyl chain 
> substituent can lead the compound to achieve nM affinity (>10-fold better 
> than any other substituent) — in particular the binding site is not 
> hydrophobic and no interaction is found between the substituent and the 
> protein.
>
> Anything I have miss here that can increase the binding affinity without 
> interacting with the target?
>
> Thanks.
>
> Kind regards,
> Wenhe
>
>
>

[ccp4bb] Compound with flexible conformation but nM Kd

[WENHE ZHONG]

Dear Community,

A little bit out of topic here. We are applying the structure-based approach to 
design compounds that can bind our protein target. We have synthesized a series 
of analogues based on the same scaffold with different substituents at one 
particular site. The most potent analogue (nM Kd) has a long alkyl chain 
substituent. We thought this hydrophobic substituent should have strong 
interactions with the target protein leading to nM range affinity. However, 
crystal structures show very weak densities for this substituent and no obvious 
interaction between the substituent and the target protein, suggesting that 
this long alkyl chain substituent is flexible without binding to the protein. 
This binding site is relatively negative charged according to the electrostatic 
potential analysis. 

So it is a puzzle to me that how this dynamic and hydrophobic alkyl chain 
substituent can lead the compound to achieve nM affinity (>10-fold better than 
any other substituent) — in particular the binding site is not hydrophobic and 
no interaction is found between the substituent and the protein.

Anything I have miss here that can increase the binding affinity without 
interacting with the target?

Thanks.

Kind regards,
Wenhe